Many modern techniques of biochemistry and biotechnology are based on the analysis of biological particles, e.g. cells. Several parameters concerning the type or species of the particles, as well as the state of the particles, such as viability, are among parameters and properties that are routinely investigated. Further information about intercellular status is also frequently determined. In this field, the method of luminescence detection, e.g. fluorescence detection, has gained a wide application, mainly due to its inherent specificity and sensitivity.
Photoluminescent analysis of material, such as biological material, is based on illuminating a sample with light (excitation) of a given wavelength and collecting light emitted (emission) from the sample, parts or components of the sample, at another substantially different wavelength. The difference in wavelength between excitation and emission (generally called Stoke's shift) is a property of the sample being analysed, more generally a property of the photoluminescent molecules present in the sample. If the Stoke's shift is great enough to allow substantial optical separation of excitation and emission light, it is feasible to use the method of photoluminescence for the analysis of the material.
The photoluminescent emission (e.g. phosphoresce or fluorescence) is typically low in intensity compared to the excitation light, usually by the order of several magnitudes. The fact that emission is detected against “darkness”, makes the method well suited, since many of the detectors commercially available show low response to “darkness” while responding considerably well to light, e.g. photons, striking the detector. Nevertheless improved sensitivity, e.g. expressed as increased emission, is typically a favourable property and therefore there exist currently numerous methods for that purpose in the prior art.
Increase in the intensity of excitation light generally increases the amount of emitted light, since the probability of generating emission is proportional with the number of photons interacting with photoluminescent molecules. One often used method for the increase of excitation intensity is the use of laser, which are available in configurations where the amount of emitted light is strong, both since the amount of emitted light, e.g. expressed as energy flux, is considerable, but also since the light from a laser is easily focused onto a small area, thus generating high light density, e.g. expressed as emitted energy per area.
Another method for illumination of photoluminescent material is to use a dispersed light source, such as a lamp or a light emitting diode. The advantage of such light sources is that they emit dispersed light, thus allowing the illumination of a considerably large area. One common drawback to these light sources has typically been the relatively poor degree of homogeneous illumination of the sample material, obtained simultaneously with high degree of efficiency, defined by the fraction of emitted light striking the sample material.
Often preferred equipment for biological analyses is a microscope, typically equipped with two or more objectives for variable magnifications. Further, fluorescence detection requires a wavelength specific excitation and emission filters. The operation of a microscope has to some degree been automated, mainly through the implementation of image analysis. However, even with such automation, the operation of a microscope is primarily a manual task, requiring considerable training of the operator.
Automated flow cytometers are also used for analyzing particles such as cells. Flow cytometer is a synonym for a wide range of equipment characterised by analysing particles under flow conditions, where these conditions usually allow individual particles to be analysed one at a time. Flow cytometer in certain versions make complex analyses of biological particles available, but flow cytometers are difficult to use because the operation usually requires considerable operator skill.
The apparatus and method of the present invention also addresses the areas of cell viability. The determination of cell viability is important for assessing the effects of e.g. drugs, environmental pollutants, temperature, ionic extremes and radiation on cells and tissues. Cell membrane integrity is commonly used as indicator of cell viability. A feature of loss of membrane integrity is the formation of pores that permit the passage of low molecular weight molecules (MW<2000 Daltons) in and out of the cell. The enhanced permeability has been the basis for many cell viability assays. The most common methods used for viability measurements are 51Cr release, Trypan blue exclusion and the combination of different fluorescent dyes to detect live and dead cells. U.S. Pat. No. 6,403,378 describes a method based on membrane integrity that utilizes two fluorescent dyes, one which labels all dead cells with compromised membranes while the other labels all living cells. To obtain reliable results for different cell populations and densities using a two-dye method it is crucial to carefully control the amount of each dye and the incubation time used to stain the cells. Propidium iodide and ethidium bromide are excluded from the cytosol, and hence the nucleus, of viable cells and are mentioned as the most common fluorescent tracers for staining dead cells. In contrast, acridine orange and Hoechst-33342 are readily taken up by viable cells and are often used as fluorescent probes for staining living cells.
Acridine orange (IUPAC name: N,N,N′,N′-tetramethylacridine-3,6-diamine, synonyms: Basic Orange 3RN, Euchrysine, Acridine Orange NO, Rhodulin Orange NO, Waxoline Orange etc.) is a nucleic acid selective fluorescent cationic dye that interacts with DNA and RNA by intercalation or electrostatic attractions. When bound to double stranded DNA and RNA acridine orange has excitation maximum at 502 nm and an emission maximum at 525 nm. When it associates with single stranded nucleic acid, the excitation maximum shifts to 460 nm and the emission maximum shifts to 650 nm. Acridine orange is also known to show alterations of absorbance and fluorescence properties in its different forms. The monomeric dye in solution exhibits a green fluorescence, whereas the stacking of acridine orange in oligomeric structures will have a red emission (Kapuscinski et al., 1982: Luminescence of the solid complexes of acridine orange with RNA. Cytometry 2, pp. 201-211). This alteration results from a concentration-dependent increase in resonance energy transfer among individual acridine orange molecules, and increasing concentrations of acridine orange in a solution will induce progressive quenching of the green emmission (Millot et al., 1997: Characterization of Acidic Vesicles in Multidrug-resistant and Sensitive Cancer Cells by Acridine Orange Staining and Confocal Microspectrofluorometry. The Journal of Histochemistry & Cytochemistry 45(9): pp. 1255-1264). Acridine orange will also enter acidic compartments such as lysosomes and become protonated and sequestered. In these low pH conditions, the dye will emit red light when exited by blue light. In conclusion, this shows that the fluorescence emission spectrum of acridine orange is affected by many factors, including the gross secondary structure of the polynucleotides, pH and the concentration of acridine orange.
DAPI or 4′,6-diamidino-2-phenylindole is another fluorescent dye that has been described as cell permeable and useful for staining of living cells (e.g. Betty I. Tarnowski; Francis G. Spinale; James H. Nicholson. 1991. Biotechnic and Histochemistry, 66: 296-302). However, careful studies in our laboratories have revealed that DAPI penetrates cells with a rather slow kinetics. Thus, by controlling the concentration and incubation time DAPI can be used as a probe for staining nonviable cells and can therefore be used to discriminate between live and dead cells. DAPI preferentially binds to double stranded DNA and associates with AT clusters in the minor groove. When bound to double-stranded DNA its absorption maximum is at 358 nm and its emission maximum is at 461 nm. Binding of DAPI to DNA produces a 20-fold fluorescence enhancement. DAPI will also associate to RNA, though in a different binding mode. The emission peak of the DAPI/RNA complex is red-shifted to around 500 nm and the quantum yield is only 20% of that of the DAPI/DNA complex.
The combination of acridine orange and DAPI has not previously suggested been as or demonstrated to be suitable for a simultaneous or two-color fluorescence assay of cell viability.
The apparatus and method of the present invention also addresses the areas of transfection. Transfection, the introduction of foreign nucleic acid (DNA or RNA) into a eukaryotic cell, is a common and important laboratory procedure for studying the gene and protein function in living cells. There are numerous methods available for cell transfection such as formation of complexes of the nucleic acid with either DEAE dextran or calcium phosphate to allow cell uptake by endocytosis, or eletroporation, which employs pulses of voltage to form pores in the plasma membrane through which the nucleic acid can enter. Most transfection procedures, however, involve complexes of nucleic acids and cationic lipids followed by fusion with cells and delivery of DNA/RNA into the cytosol. While rather routine, transfection requires optimization of assay conditions for different cell types. There are a variety of methods for determining transfection efficiency in cell populations. Most of these monitor the expression of a fluorescent, luminescent or colorimetric reporter gene. The reporter gene can be present on the same vector as the gene of interest or on a separate vector. Convenient reporter genes for measuring transfection efficiency is autofluorescent proteins, e.g. green fluorescent protein (GFP) isolated from the jelly fish Aequorea victoria and red fluorescent protein (RFP) developed from the marine anemone Discosoma striata, both of which enable assays on living cells and requires no substrate for generation of fluorescence. When excited by blue light, GFP emits green light, whereas RFP emits orange/red light when excited with green light. Moreover, the combination of GFP with appropriate dyes allows multiplex analysis to estimate e.g. viability, cytotoxity and apoptosis.
Another approach for monitoring transfection efficiency employs fluorescently labeled nucleic acids as reporter, e.g. Cy5-labeled siRNA to optimize RNAi silencing experiments.
In one method of analysis, the GFP transfected cells are incubated with DACM and propidium iodide (PI). DACM reacts with thiols, the level of which is low in dying/dead cells, to produce a blue fluorescent product in living cells. In contrast, PI only penetrates cells with damaged membranes and, thus, only labels DNA of dead cells. Cells labeled with DACM are detected by UV/violet excitation and measuring blue light, whereas PI labeled cells are detected by green light excitation and measuring the emitted red light. Cells expressing GFP (transfected cells) are monitored by blue light excitation and measuring green light. Information about transfection efficiency and e.g. viability can be extracted from the data.
In another method of analysis, the RFP transfected cells are incubated with DACM and acrdine homodimer. Acridine homodimer only penetrates cells with damaged membranes and, thus, only labels dead cells. Living cells labeled with DACM are detected by UV/violet excitation and measuring blue light, whereas dead cells labeled with acridine homodimer are detected by blue light excitation and measuring the emitted green light. Cells expressing RFP (transfected cells) are monitored by green light excitation and measuring red light. Information about transfection efficiency and e.g. viability can be obtained from the data.
In a third method of analysis, cells transfected with siRNA, labeled with a green fluorophore, e.g. FITC, are incubated with DACM and PI. Living cells labeled with DACM are detected by UV/violet excitation and measuring blue light, whereas dead cells labeled with PI are detected by green light excitation and measuring the emitted red light. Cells harboring siRNA (transfected cells) are monitored by blue light excitation and measuring green light. Information about transfection efficiency and e.g. viability can be pulled out from the data.
The combination of a thiol-reacting reagent and a cell impermeable DNA stain in a cell population transfected with nucleic acid has not previously been suggested or demonstrated to be suitable for a simultaneous assay of transfection efficiency, cell viability and cytotoxity.
The apparatus and method of the invention also addresses the area of cell cycle. The cell cycle represents the most fundamental and important process in eucaryotic cells. An ordered set of events, culminating in cell growth and division into two daughter cells, the cell cycle is tightly regulated by defined temporal and spatial expression, localization and destruction of several cell cycle regulators. Cyclins and cyclin-dependent kinases (CDK) are major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to M phases. In a given population, cells will be distributed among three major phases of cell cycle: G1/G0 phase (one set of paired chromosomes per cell), S phase (DNA synthesis with variable amount of DNA), and G/M phase (two sets of paired 2 chromosomes per cell, prior to cell division).
The most common approach to determine the cell cycle stage is based on measurement of cellular DNA content. DNA content can be determined using fluorescent, DNA-selective stains that exhibit emission signals proportional to DNA mass. Cellular fluorescence is measured by flow, image or laser scanning cytometry. This analysis is typically performed on permeabilized or fixed cells using a cell-impermeant nucleic acid stain, but is also possible using live cells and a cell-permeant nucleic acid stain.
Because cell cycle dysregulation is such a common occurrence in neoplasia, the opportunity to discover new targets for anticancer agents and improved therapeutics has been the focus of intense interest. The cell cycle assay has applicability to a variety of areas of life science research and drug development, including cancer biology, apoptosis analysis, drug screening and measuring health status of cell cultures, e.g. in bioreactors.
DAPI is a competent dye for measurement of the cell cycle stage. However, excitation of DAPI requires a UV light source and standard flow cytometers usually come without a UV light source hampering the use of DAPI.
The apparatus and method of the invention also addresses the area of cell death. Cell death may occur by two distinct mechanisms, necrosis or apoptosis. Necrosis occurs when cells are exposed to harsh physical or chemical stress (e.g., hypothermia, hypoxia) while apoptosis is a tightly controlled biochemical process by which cells are eliminated and where the cell is an active participant in its own termination (“cellular suicide”). Apoptosis is one of the main types of programmed cell death which occur in multicellular organisms and is characterized by a series of events that lead to a variety of morphological changes, including blebbing, nuclear fragmentation, chromatin condensation, cell shrinkage, loss of membrane asymmetry and translocation of the membrane phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane.
Apoptosis is both a very complex and very important process and dysregulations in the apoptosis machinery may lead to very severe diseases. A growing body of evidence suggests that resistance to apoptosis is a feature of most, if not all types of cancer. Moreover, defects in apoptosis signaling contribute to drug resistance of tumor cells. In the other hand may hyperactivity of the apoptotic processes also cause diseases such as neurodegenerative diseases as seen in Parkinson's or Alzheimer's Diseases, where apoptosis is thought to account for much of the cell death and the progressive loss of neurons.
As apoptosis play a very important role in a wide array of biological processes, including embryogenesis, ageing, and many diseases, this type of programmed cell death is the subject for many studies, and tools for easy detection and investigation of apoptosis are desirable.